Changes in lipid metabolism driven by steroid signalling modulate proteostasis in C. elegans

Abstract Alzheimer's, Parkinson's and Huntington's diseases can be caused by mutations that enhance protein aggregation, but we still do not know enough about the molecular players of these pathways to develop treatments for these devastating diseases. Here, we screen for mutations that might enhance aggregation in Caenorhabditis elegans, to investigate the mechanisms that protect against dysregulated homeostasis. We report that the stomatin homologue UNC‐1 activates neurohormonal signalling from the sulfotransferase SSU‐1 in ASJ sensory/endocrine neurons. A putative hormone, produced in ASJ, targets the nuclear receptor NHR‐1, which acts cell autonomously in the muscles to modulate polyglutamine repeat (polyQ) aggregation. A second nuclear receptor, DAF‐12, functions oppositely to NHR‐1 to maintain protein homeostasis. Transcriptomics analyses of unc‐1 mutants revealed changes in the expression of genes involved in fat metabolism, suggesting that fat metabolism changes, controlled by neurohormonal signalling, contribute to protein homeostasis. Furthermore, the enzymes involved in the identified signalling pathway are potential targets for treating neurodegenerative diseases caused by disrupted protein homeostasis.


Isolation of the unc-1(vlt10) mutation
We induced random mutagenesis using 47 mM EMS (methanesulfonic acid ethyl ester, Sigma, St. Louis, Missouri, USA) in L4 animals of the AM141 strain. We incubated worms for 4 h at 20°C on this solution. After washing them they were pipetted onto NGM plates, seeded with OP50, and allowed them to lay the F1. F1 animals were bleached when they reached adulthood, and we searched among the F2 for animals uncoordinated and with abnormal aggregation patterns. Once isolated animals with the right phenotype (i.e. with abnormal motility and altered polyQ aggregation), they were outcrossed 5 times against the wild type background (N2, Bristol), before high-throughput sequencing of their genomic DNA. The reads provided by the sequencing service (Centre Nacional d'Anàlisi Genòmica -Centre de Regulacio Genomica, Barcelona, Spain) were mapped against the C. elegans WS245 reference using the mem algorithm implemented by the BWA software (Li, 2011(Li, , 2013. BAQ qualities were calculated and applied to the BAM alignments by using samtools calmd (Li, 2011). The SNP calling process was carried out by Freebayes (Garrison & Marth, 2012) with a minimum mapping quality of 57, a base quality threshold of 20, a minimum coverage of 6 and a minimum SNP quality of 20. A filtering process was stablished to look for the SNPs whose allelic frequencies were likely to have been affected by the selection process. SNPs were filtered out if they had more than one allele in all families, or if the reference allele was not present in any family, or if there were more than three mutant alleles when all families were considered. For the SNPs that passed all the filters a selection index was calculated. It consisted in the difference between the frequency of the most frequent allele in the back-crossed population and the frequency of that same allele in parental population, thus the SNPs with the highest differences between both populations would had the highest selection index. The predicted effect of each SNP was calculated by SnpEff (Cingolani et al, 2012).
To determine the molecular identity of this allele we sequenced the whole genome of this strain after six outcrossing steps and also the genome of the original strain (RVM10). EMS causes random lesions through the chromosomes and after outcrossing RVM10, most of these mutations would be lost during outcrossing, except the DNA changes that lie around the allele responsible for the aggregation phenotype. We took advantage of this, and when we compared both genomes we observed a region with dense amount of mutations that "peaked" around the left arm of chromosome X (Appendix Figure S1A). Detailed analysis of this region showed that the unc-1 gene, of RVM10, had a nonsense mutation in homozygosis that gives rise to premature stop codon, which would produce a putative null (Appendix Figure S1B). unc-1 encodes a homologue of the Stomatin-like protein family from mammals (Lapatsina et al, 2012;Rajaram et al, 1998).
The product of this gene has been shown to be involved in modulation of the electrical synapse (Chen et al, 2007) and sensitivity to anaesthetics (Rajaram et al, 1998). Null alleles of this gene are well-known to cause strong uncoordination (Chen et al, 2007), similar to vlt10 mutant from our screen. The double band identified by the anti-polyQ antibody most likely reflects proteolytic cleavage of 40Q::YFP. We used actin to normalize loading levels. (D) ImageJ quantification of 40Q::YFP protein levels in all genetic backgrounds, normalized to 40Q::YFP wild type (grey) to verify any changes according to transgene expression. All plotted data show the mean ± standard error of the mean (SEM). We performed ANOVA test, with post-hoc Tukey test, which shows that there were not significant differences between samples. Three biological samples were evaluated for each strain. Thirty animals per condition and/or strain and per experiment were analysed. Each analysis has been reproduced at least three times. ***P < 0.001; **P < 0.01; ns: not significant in reference to the wild type 40Q::YFP strain (graph A), as calculated using the one-way ANOVA with posthoc Tukey test.  Data information: n = 15 (wild type), 17 (unc-1(vlt10) animals, recorded over at least three days. ** P < 0.01, ns: not significant, as calculated using unpaired t-test. Appendix Figure S5. Phylogenetic analysis of C. elegans and human sulfatases. The diagram shows the phylogenetic tree of all C. elegans sulfatases (SUL-1, SUL-2 and SUL-3) obtained using the MUSCLE (Edgar, 2004) and IQ-TREE version 1.6.8 software (Chernomor et al, 2016;Li, 2013;Nguyen et al, 2015). The phylogenetic tree shows bootstrap values which provides confidence values for each node. This analysis suggests that SUL-2 and SUL-3 are closer to arylsulfatases, while SUL-1 is closer to hSULF1 and hSULF2.

( R N A i ) r a b -3 p : : i n x -6 ( R N A i ) r a b -3 p : : i n x -2 ( R N A i ) r a b -3 p : : u n c -7 ( R N
Appendix Figure S6. NHR-1 regulates the expression of some genes related to DAF-12 signalling. (A) Graph of the relative expression of daf-36, a gene that encodes an oxidoreductase enzyme involved in cholesterol processing, in unc-1 and nhr-1 single and double mutants compared to wild type worms. (B) Graph of the relative expression of daf-9, a cytochrome involved in dafachronic acids synthesis, in unc-1 and nhr-1 single and double mutants compared to wild type worms. (C) Diagram of parts of the synthesis pathway of dafachronic acids, placing the activity of the HSD-1, DAF-36 and DAF-9 enzymes. Data information: The plotted data show mean ± standard error of the mean (SEM). Six biological replicates were analysed for each strain. **P < 0.01; ***P < 0.005, as calculated using the one-way ANOVA with post-hoc Tukey test and compared to unc-1 mutants (2) unc-1_vs_nhr-1 For example, graph D shows that there are 523 genes that are differentially expressed between double and 40Q; unc-1 mutants and also between unc-1 and 40Q, but not between double mutants and 40Q. This shows that these genes are rescued by the vlt16 allele. In contrast, there are 173 differentially expressed genes that are specific to animals carrying the vlt10 allele and that are not altered by vlt16.
Appendix Figure S8. Diagram showing the production of PCR-generated constructs to induce RNAi. A promoter sequence for a gene fragment was assembled to promote tissuespecific RNAi via fusion PCR. External (ext) and internal (int) primers were used to amplify the promoter region (red), sense (purple) and antisense (blue) sequence from genomic DNA. The primers had 20 complementary nucleotides (red tail on primers) that allow fusion using nested primers.  Appendix Figure S9. CRISPR strategies to modify gene expression. (A) The inx-2 gene was disrupted using two sgRNAs that cut all coding sequences of the gene. The vlt22 allele obtained from this experiment represents a complete loss of the inx-2 gene. (B) The daf-12 gene was disrupted using a combination of two gRNAs that target exons 12 and 14. vlt19 is a 500-bp deletion that affects the ligand-binding domain (LBD) in the C-terminal region of the DAF-12 protein. (C) Diagram to introduce the vlt16 mutation in nhr-1, which emulates the allele n6242 and the novel vlt15 allele as a result of an abnormal homologous recombination containing duplicate insertions (yellow and blue shading). vlt15 induces a frameshift (red pattern) that produces a change (C/T) and a premature TGA stop codon upstream. Both alleles, vlt16 and vlt15, are putative nulls.